Decoder-seq: a technology for high sensitivity, high resolution, and low-cost spatial RNA sequencing

The spatially resolved transcriptomics (SRT) technology serves as a powerful tool for delineating the spatial gene expression patterns of tissues, revealing cellular composition, distribution and interactions. Its applications span across various research fields such as embryonic development, neuroscience, and diseases, attracting widespread attention. Existing SRT technologies mainly include: (1) imaging-based in situ hybridization or sequencing (Chen

increases sequencing costs to some extent.Therefore, the current spatial barcoded array-based sequencing technology faces challenges in balancing gene detection sensitivity and spatial resolution.The complex process of preparing barcoded DNA arrays and their high cost also pose certain technical barriers for many researchers and communities.
A recent study by Cao et al. established an assay called reported Decoder-seq (Cao et al. 2024), which is a flexible and accessible spatial barcoded array-based sequencing technology that enables high sensitivity, high resolution and low-cost SRT analysis (Fig. 1).By assembling spherical dendrimers on glass slides, 3D nanostructured substrates with abundant amino functional groups were formed.This significantly enhances the modification density of spatial DNA barcodes by about an order of magnitude compared to other substrates, while also increasing the distance between barcodes and substrates, thereby improving accessibility for effective mRNA capture.To streamline the fabrication of a barcoded DNA array, a microfluidicsbased barcoding strategy was employed to generate deterministic and combinatorial X i Y j coordinates on 3D dendrimeric substrates.A pair of microfluidics chips with channels perpendicular to each other were designed and sequentially placed on the slide to introduce X and Y sets of barcode solutions.By adjusting the number and width of the microchannel, the barcoded DNA arrays with different capture areas and spatial resolutions (50, 25, 15 and 10 μm) were flexibly generated.Moreover, this deterministic combinatorial barcoding strategy significantly reduces the variety of DNA barcodes without requiring decoding steps and greatly reduces costs.Compared to other similar technologies, Decoder-seq is affordable and accessible, making it easily adoptable by other labs using readily available reagents and instruments.
Using model tissues of mouse olfactory bulbs (MOBs), Cao et al. have successfully demonstrated the high gene detection sensitivity of Decoder-seq.The detection sensitivity of Decoder-seq was approximately (68.9 ± 15.6)% compared to in situ sequencing technology.Decoder-seq with near cellular spatial resolution (15 μm) detected 40.1 UMI and 14.7 genes per μm 2 , surpassing other cutting-edge spatial barcoded arraybased sequencing technologies.Compared to commercial 10× Visium, Decoder-seq identified a five-fold increase in lowly-expressed olfactory receptor (Olfr) genes.More importantly, the 15 μm-spot Decoder-seq observed a unique layer enriched spatial patterns of two Olfr genes that were not discernible using other similar methods.This indicates that the high gene detection sensitivity is advantageous for spatial localizing of lowly-expressed genes, such as Olfr, enabling analysis of previously challenging basic physiological activities like olfactory mechanism in a high-throughput manner.Besides, based on an imagebased cell segmentation algorithm and cell-type deconvolution, Decoder-seq successfully generated a spatial single-cell atlas of the mouse hippocampus that faithfully aligned with the ground truth of the HE image.Finally, Decoder-seq investigated the spatial heterogeneity and potential tumor invasion behaviors of two subtypes of human renal cell carcinoma (RCC), predicting clinical outcomes of RCC patients by identifying a panel of gradient-expressed epithelial-mesenchymal transitionrelated genes.This highlights the potential of Decoderseq for analyzing complex tissue samples.
In sum, the establishment of the Decoder-seq platform offers unique opportunities for researchers to understand the basic biological principles as well as deep pathological mechanisms.The fabrication process of barcoded DNA arrays is characterized by its simplicity and cost-effectiveness, making it replicable and affordable for adoption of the Decoder-seq technology in

Fig. 1
Fig. 1 Workflow of Decoder-seq referring from Cao et al. (2024).A Generation of a 3D spatial barcoded DNA array by combining 3D dendrimeric substrate with microfluidics-based combinatorial barcoding strategy.B Spatial transcriptome indexing.C SRT analysis through bioinformatic Open Access This article is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.